Talk Transgenic Technologies · Transgenic animals For what reason? Transgenesis in Mice Retroviral...
Transcript of Talk Transgenic Technologies · Transgenic animals For what reason? Transgenesis in Mice Retroviral...
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Transgenic animals
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Transgenic animals
• For what reason?• How to do it...• Can you clone an animal?• How can you use it?
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Transgenic animalsWhy?
• Selective breeding is performed since centuries.
• Breeding is time consuming.• Crossing in properties is time consuming.• Only a limited number of properties
available.
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• Introduction of a desired property(hypothesis driven; without hypothesis)
• Fast generation of animal lines carrying the desired property
• Animal model for human diseases• Animal system to produce biomolecules
(„Pharming“)• Xeno-Transplantates
Transgenic animalsFor what reason?
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Transgenesis in MiceRetroviral Vector
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• Efficient mechanism of transgene integration • Transfer of genes < 8 kb possible• random insertion of the transgene• Retroviral contamination of the transgenic
animal
Transgenesis in MiceRetroviral Vector
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Transgenesis in MiceMicroinjection
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Transgenesis in MiceMicroinjection
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Transgenese in MäusenMikroinjektion
• Low efficient mechanism of transgeneintegration (5-10%)
• Transfer of large genes possible• random insertion of the transgene• Variable expression levels
no selection of the transgene necessary
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Efficiency of Microinjection
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GFP under the CMV Promoter
Transgenesis in Mice
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Transgenesis in MiceStem cell Method
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Embryonic Stem cells~30 cells 200-250
cells
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Embryonic Stem cells
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Stem cell differentiation
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Gene Knockout
Target Vector
Chromosome
Chromosome
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Negative Selection
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Positive Selection
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Vasectomia of male mice
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Blastocysts
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Injection of embryonic stem cells
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Implantation of Blastocysts
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3
Implantation of Blastocysts
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Black mouse -no ES cell integration
Chimeric mouse -high ES cell integration
Chimäre Mäuse
Chimeric mouse -low ES cell integration
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Cross breeding strategies
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• Efficient transfection• positive-negative Selection • Targeted insertion of the gene• Characterization before animal
generation• Time and cost intensive
Transgenesis in MiceStem cell Method
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1 2 3
lacZ-tagged insertion allele
FRTFRT
loxPloxPβgalneo
lacZ-tagged null allele (Δ exon)
1 βgal 3FRTFRT
loxP
Cre recombinase
βgalneo
pre-conditional allele (wild-type)
1 2 3
null allele (Δ exon, frameshift, NMD)
Flp recombinase
1 3
Cre recombinase
Transgenesis in Mice“Smart“ recombination Methods
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“Knock-in” mouse model fornephrogenic Diabetes insipidus
loxP loxPE242X
K.O.
WildtypeK.O.
Exon 1 Intron 1 Exon 2 NeoR Exon 3/3’-UTR
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Pathomechanism of nephrogenic Diabetes insipidus
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Mouse Model of the X-linked NDI
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ß-galactosidase under the Neurogenin promoter
Transgenesis in miceUse
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Cloning by nucleus transfer
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„Dolly“• 1997, first living
offspring derived from a differentiated cell
• Is Dolly a real clone?
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„Gene“
• 1997, first living bovine offspring derived from a stem cell of a 30 days-old fetus
• Difference compared to Dolly?
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„Cc:“• First clone cat
• Derived from fibroblast cell by nucleus transfer.
• Coat color differs between the genetic mother and the offspring
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Commercialized pet cloning
• available: Cryoconservation of cellscosts: $895 - $1395
• Texas A&M takes $250,000 for cloning a cat
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Identification of transgenic Mice